Evaluating Nutraceuticals for Selective Toxicity Toward Leukemia Stem Cells

Abstract

Targeting leukemia stem cells (LSCs) is critical to improving the poor outcome of acute myeloid leukemia (AML) patients. Nutraceuticals (i.e., food derived bioactive compounds) provide a wealthy resource for novel anti-cancer, and specifically anti-AML drug discovery. With the advent of novel LSC cell lines, preliminary screening of these compounds against LSC-like cells can be achieved rapidly. To identify potential novel anti-LSC therapeutics, we created and screened a unique library consisting of 288 nutraceuticals in an MTS assay against TEX leukemia cells, a surrogate LSC line and K562, a control cell line which does not possess LSC activity. Here, we identified diosmetin, a flavonoid found in citrus fruits and various green plants, as a novel anti- LSC agent (EC50: 6.0 ± 1.7μM). To confirm its activity, diosmetin (10μM) reduced clonogenic growth of primary AML cells (n = 4) with no effect on normal CD34 positive bone marrow derived stem cells (n = 3) observed in colony forming cell assays. A dose-response and time course analysis performed via the Annexin/PI assay and flow cytometry revealed that diosmetin induced apoptosis, as evidenced by the accumulation of ANN+/PI- cells. Apoptosis was further confirmed by a subG1 peak after performing cell cycle analysis. Utilizing the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool, we determined that the estrogen receptor (ER) was a potential molecular target for diosmetin’s anti-leukemia activity. To assess the role of estrogen receptors, we measured ERα and ERβ protein levels in diosmetin sensitive and insensitive cell lines. Interestingly, diosmetin sensitive cell lines display significantly elevated ERβ protein levels compared to diosmetin insensitive cells. However, this pattern was not observed for ERα. Similar results were observed through quantitative PCR measures, as TEX cells displayed levels of ESR2 (ERβ) mRNA, with no observed levels of ESR1 (ERα) mRNA levels. The opposite results were observed in K562 cells. Through ER reporter assays, it was demonstrated that diosmetin acts as a partial agonist in ERβ reporter cells, increasing luciferase activity with increasing doses of diosmetin in ERβ reporter cells. Moreover, we find that caspase 8 but not caspase 9 is elevated following diosmetin treatment, consistent with the extrinsic pathway of apoptosis and our observed increased in TNF-α, similar to previous reports highlighting the link between ERβ agonists and cancer cell death. In summary, these studies highlight that estrogen receptors, specifically ERβ, is a novel LSC therapeutic target, and the potential role of nutraceuticals as promising compounds for future drug discovery endeavours.